Assay for the diagnosis of macular degeneration

ABSTRACT

An assay and kit for detecting N-retinylidene-N-retinylethanolamine (A2E) in a sample that uses a lipid binding protein, such as Saposin B, to capture A2E in the sample and assist in the extraction and measurement of A2E via mass spectroscopy. A2E thus serves as a marker for macular degeneration so that the assay and kit of the invention can be used to detect the presence or severity of macular degeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional App. No.62/412,501, filed on Oct. 25, 2016.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to the diagnosis of eye disease and, morespecifically, to an assay for detecting toxic eye compounds in urine.

2. Description of the Related Art

The human eye is made up of different tissues that include conjunctiva,cornea, pupil, iris, aqueous humor lens, ciliary body, vitreous humor,retina, choroid, sclera, optic nerve and muscles. The outermost shell oftissue is a layer of transparent membrane called the conjunctiva. Itcovers a white layer of tissues called the sclera. The cornea isattached to the sclera in front of the eye. Light passes through thecornea into the pupil, which constricts or dilates to control the amountof light that enters the eye. Pupil is in the center of the iris, whichis outside of the lens. The change of the size of the pupil is madepossible by the ciliary muscles surrounding the pupil. Between the lensand the cornea, there is a kind of liquid called the aqueous humor.Light travels through cornea and pupil, focuses on the lens and thenonto the retina, which is a layer of sensitive light-sensing cells. Theretina then converts the light signals into electrical signals andpasses them onto the visual cortex where the signal is translated intoimages for the brain to analyze. The macula is the center of the retina.A small depression is present at the center of the macular where thelight focuses and forms the clearest vision.

Macular degeneration (MD) is an age related eye disease that is markedby the loss of vision in the center of the eye. It usually happens toolder people and is the major cause of vision loss and blindness amongthem. There are two forms of MD, the dry form and the wet form. The dryform of macular degeneration is an initial, less serious form, and iscaused by the gradual loss of photoreceptors of the macula. Thisphotoreceptor loss is thought to arise from the accumulation ofintracellular and extracellular material within the eye. Intracellularaccumulations, termed lipofuscin, are found within essential supportcells called retinal pigmented epithelial (RPE) cells in the macula. TheRPE cells are important in that they support the light sensitivephotoreceptor cells. The failure of RPE cells leads to death ofphotoreceptors and a progressive loss of vision. Extracellularaccumulations, termed drusen, increase in size and quantity as MDprogresses. Lipofuscin mediated RPE cell death is thought to contributeto drusen formation. As drusen accumulates, it can destabilize themacular region by contributing to inflammation, complement activation,and other processes. Over time, dry MD can progress to the more advancedwet form of macular degeneration, also referred to as neovascularmacular degeneration. The neovascular macular degeneration occurs whenabnormal blood vessels from the vascular layer of the eye enlarges.These blood vessels have the potential rupture, spilling blood and fluidinto the retina. Serious complications including blindness can ensue.

Significant lipofuscin that accumulates with age and in certaindisorders of RPE cells is the result of disregulation of vitamin Arecycling. Major lipofuscin constituents include the di-retinalconjugate N-retinylidene-N-retinylethanolamine (A2E) and itsphotoisomers, which have adverse effects due to their amphiphillicityand photoreactivity. Excessive lipofuscin accumulation and AMD areconsidered strongly correlated. Currently, there is no assay availablethat can detect the presence or severity of macular degeneration.Accordingly, there is a need in the art for an approach that can detectthe presence of indicators like A2E and thus detect the presence orseverity of macular degeneration.

BRIEF SUMMARY OF THE INVENTION

The present invention is an assay for detectingN-retinylidene-N-retinylethanolamine (A2E) in urine. The lipid bindingprotein Saposin B is used to capture A2E in a sample, such as a blood orurine sample. The captured A2E may then be extracted from the urineusing immunoprecipitation and measured via mass spectroscopy. Thepresent invention thus includes a kit for performing an assay for thediagnosis of macular degeneration, comprising a quantity of a lipidbinding protein and an immunoprecipitation substrate having a pluralityantibodies to the lipid binding protein. The lipid binding protein maybe Saposin B. The immunoprecipitation substrate may comprise protein Gbeads that have been incubated with antibodies targeting Saposin B. Theantibodies may be IgG anti-saposin B antibodies.

The present invention may also comprise a method of diagnosing a subjectas having macular degeneration, comprising the steps of obtaining asample from the subject, adding a quantity of a lipid binding protein tothe sample to bind any N-retinylidene-N-retinylethanolamine in theurine, and incubating the urine sample and the quantity of the lipidbinding protein with an immunoprecipitation substrate having a pluralityof antibodies to the lipid binding protein. The method may also comprisethe step of collecting any bound lipid binding protein andN-retinylidene-N-retinylethanolamine. The method may additionallycomprise the step of determining how much of any bound lipid bindingprotein and N-retinylidene-N-retinylethanolamine has been collected. Thestep of determining how much of any bound lipid binding protein andN-retinylidene-N-retinylethanolamine has been collected may comprise theuse of a mass spectrograph

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1A is a first fluorescence spectroscopy graph showing that SaposinB binds A2E;

FIG. 1B is a second fluorescence spectroscopy graphs showing thatSaposin B binds A2E;

FIG. 2 is a mass spectrum of A2E; and

FIG. 3 is a mass spectrum of A2E recovered from SapB-A2E in urine.

FIG. 4 is a Western blot of SapB from urine, RPE cells, and E. coliexpression where the lanes (from left to right) are: Lane 1, Gel Ladder;Lane 2, Saposin B from urine; Lane 3, Retinal Pigment Epithelium cellSaposin B; Lane 4, RPE cell Saposin B; and Lane 5, Saposin B expressedin BL21 E. coli.

DETAILED DESCRIPTION OF THE INVENTION

Referring to the figures, wherein like numerals refer to like partsthroughout, the present invention comprises an assay that employssaposin B (sapB) to bind A2E in human urine so that is may be detectedas measured as an indicator for the presence and severity of maculardegeneration. A2E is a compound produced as a part of the light cycle inthe human eye and is associated with the development and/or progressionof macular degeneration. As A2E builds up in the eye, it is possible itis removed from the body via the blood and then the kidneys in urine,with levels rising in the blood and urine as the disease progresses.Thus, the present invention uses a lipid binding protein, such asSaposin B, to bind A2E, for extraction and measurement.

Example

SapB bound A2E was pre-complexed in a 2:1 molar ratio of sapB:A2E in 50mM phosphate buffer. A volume of this solution was then added to analiquot of human urine. The sapB-A2E plus urine mixture was then addedto magnetic Protein G Beads, which had been incubated with IgGanti-saposin B antibody. After one hour of incubation at 37° C., thesample was magnetized and the excess sample volume was discarded. Thesample was then washed with 50 mM phosphate buffered saline solutionthree times. After each wash, the solution was re-magnetized and the PBSsolution was discarded. Following the third wash, mass spectrometrygrade methanol was added to the sample and incubated at 4° C. overnight.The sample was then re-magnetized and the MeOH fraction, containing theA2E, was collected for mass spectrometry analysis.

FIG. 2 depicts the mass spectrum for pure A2E and FIG. 3 shows the massspectrum for sapB-A2E recovered from the urine sample. SapB binding tothe Protein G Bead-anti SapB complex was confirmed via western blotanalysis, as seen in FIG. 4.

The present invention may comprise a kit for the extraction of A2E froma urine sample so that it can be detected and quantified that includes alipid binding protein, such as Saposin B, that can bind to A2E andenable its extraction. The kit may further include components to assistin the extraction and measurement of A2E. For example,immunoprecipitation substrates, such as protein G beads, that have beenincubated with antibodies targeting the Saposin B lipid binding protein,and the associated washing compounds, may be provided in the kit. Thepresent invention thus provides for easy and rapid quantification of aknown marker of macular degeneration.

Using A2E levels measured in the blood or urine of healthy patients,patients with diagnosed early stage macular degeneration, patients withadvancing and patients with late stage macular degeneration, arelationship between urine levels of A2E and clinical diagnosis can bedeveloped. A classic diseases progression curve or line can thus begenerated to enable physicians to track and, in early stage oncevalidated, even diagnose earlier the presence of macular degeneration.

What is claimed is:
 1. A kit for performing an assay for the diagnosisof macular degeneration, comprising: a quantity of a lipid bindingprotein; and an immunoprecipitation substrate having a pluralityantibodies to the lipid binding protein.
 2. The kit of claim 1, whereinthe lipid binding protein is Saposin B.
 3. The kit of claim 2, whereinthe immunoprecipitation substrate comprises protein G beads that havebeen incubated with antibodies targeting Saposin B.
 4. The kit of claim3, wherein the antibodies are IgG anti-saposin B antibodies.
 5. A methodof diagnosing a subject as having macular degeneration, comprising thesteps of: obtaining a sample from the subject; adding a quantity of alipid binding protein to the sample to bind anyN-retinylidene-N-retinylethanolamine in the sample; and incubating theurine sample and the quantity of the lipid binding protein with animmunoprecipitation substrate having a plurality of antibodies to thelipid binding protein.
 6. The method of claim 5, further comprising thestep of collecting any bound lipid binding protein andN-retinylidene-N-retinylethanolamine.
 7. The method of claim 6, furthercomprising the step of determining how much of any bound lipid bindingprotein and N-retinylidene-N-retinylethanolamine has been collected. 8.The method of claim 7, wherein the lipid binding protein is Saposin B.9. The method of claim 8, wherein the immunoprecipitation substratecomprises protein G beads that have been incubated with antibodiestargeting Saposin B.
 10. The method of claim 9, wherein the antibodiesare IgG anti-saposin B antibodies.
 11. The method of claim 9, whereinthe step of determining how much of any bound lipid binding protein andN-retinylidene-N-retinylethanolamine has been collected comprises theuse of a mass spectrograph.
 12. The method of claim 5, wherein thesample is a urine sample.